Title: Adrenergic Receptor Subtypes in Human Peripheral Blood Lymphocytes
Abstract: HomeHypertensionVol. 34, No. 5Adrenergic Receptor Subtypes in Human Peripheral Blood Lymphocytes Free AccessOtherPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessOtherPDF/EPUBAdrenergic Receptor Subtypes in Human Peripheral Blood Lymphocytes Annemieke Kavelaars, Charlotte Rouppe van der Voort and Cobi J. Heijnen Annemieke KavelaarsAnnemieke Kavelaars Department of Pediatric Immunology, Wilhelmina Children's Hospital of the University Medical Center Utrecht, Utrecht, The Netherlands , Charlotte Rouppe van der VoortCharlotte Rouppe van der Voort Department of Pediatric Immunology, Wilhelmina Children's Hospital of the University Medical Center Utrecht, Utrecht, The Netherlands and Cobi J. HeijnenCobi J. Heijnen Department of Pediatric Immunology, Wilhelmina Children's Hospital of the University Medical Center Utrecht, Utrecht, The Netherlands Originally published1 Nov 1999https://doi.org/10.1161/01.HYP.34.5.e5Hypertension. 1999;34:e5To the Editor:Ricci et al1 reported that human peripheral blood lymphocytes express α1-adrenergic receptor subtypes. The authors present results of radioligand binding assays with [3H]-prazosin as the radioligand for α1-adrenergic receptors, showing that the maximal density of the binding sites is 175 fmol per 106 cells. This would mean that the actual number of receptors per cell would be exactly 100 000, which is an unusually high number of receptors. Cell lines that are known for their high expression of α1-adrenergic receptors have been reported to express 10 000 to 25 000 receptors per cell.23 We have to keep in mind, however, that these numbers are found on kidney or smooth muscle cells, which are much larger than the freshly isolated lymphocytes that were used in the study by Ricci. Moreover, it is known for other G-protein–coupled receptors, eg, β2-adrenergic receptors, that are expressed on lymphocytes that the actual number of receptors per cell is around 2000.4 Thus, the number reported by Ricci et al for α1-adrenergic receptors on lymphocytes is extraordinary high.The authors do cite the literature on α1-adrenergic receptor expression in peripheral blood lymphocytes only partly correct. The only study that reports α1-adrenergic receptor binding on lymphoid cells from peripheral blood is the study by Jetschmann et al.5 However, these authors only examined NK cells where they reported the presence of 600 to 700 receptors per cell. Casale at al6 could not demonstrate binding sites on peripheral blood lymphocytes, whereas Maestroni et al7 only investigated α1-receptor expression in bone marrow cells and not in peripheral blood lymphocytes.More importantly, the authors claim that they can displace ligand from the receptor by the use of antibodies specific to the three receptor subtypes. Theoretically, this is a good approach that could give information on the presence of specific receptor subtypes. However, this approach requires antibodies that bind to the ligand-binding site of the receptor so that competition for the same site can be expected. Unfortunately, such antibodies are not available. The antibodies used in the study by Ricci et al recognize intracellular domains of the receptor and do not bind to the ligand binding domains of the α1-receptor subtypes. We have to conclude that these antibodies cannot be used in binding competition experiments, because they will not compete for binding to the same site as the ligand. In addition, it is highly unlikely that these antibodies would bind to the intracellular domains to the receptors in intact cells, since large molecules such as antibodies do not enter the cell.Another point of concern deals with the way cells are handled and the amount of cells used in the assays. In the Methods section, it is stated that 40 mL of blood was drawn from each donor. Subsequently, 10×106 cells were used for RNA isolation. The other cells were frozen at −80°. For their binding studies, it was reported that 1×106 cells per point were used and that all studies were performed in triplicate. The authors tested 9 concentrations of tritiated ligand both in the presence and absence of unlabeled ligand in order to determine aspecific binding. This part of the study would require 54×106 cells. In addition, inhibition by three different antibodies was tested, using 8 concentrations of each antibody, which would require an additional 72×106 cells. In general, with the use of 40 mL of blood, it is possible to isolate about 40×106 cells. Thus, it is not clear how all the experiments could be performed with the indicated amount of blood.When we then look at the PCR data, there is a discrepancy between the results of the RT-PCR analysis and the autoradiographic data, especially with respect to α1B. The results in panel A for α1B suggest that in lane 2, the signal is by far the strongest; whereas in panel B, this is the weakest signal.In summary, we think that in view of the technical setup of the experiments, the conclusion that freshly isolated human peripheral blood lymphocytes express α1-adrenergic receptors is not warranted by this study. References 1 Ricci A, Bronzetti E, Conterno A, Greco S, Mulatero P, Schena M, Schiavone D, Tayebati SK, Veglio F, Amenta F. α1-Adrenergic receptor subtypes in human peripheral blood lymphocytes. Hypertension.1999; 33:708–712.CrossrefMedlineGoogle Scholar2 Yang M, Büscher R, Taguchi K, Grübel B, Insel PA, Michel MG. Protein kinase C does not mediate phenylephrine-induced down-regulation of Madin Darby canine kidney cell alpha-1B adrenoceptors. J Pharmacol Exp Ther.1998; 286:36–43.MedlineGoogle Scholar3 Colucci WS, Akers M, Wise GM. Differential effects of norepinephrine and phorbol ester on alpha-1 adrenergic receptor number and surface-accessibility in DDT1MF-2 cells. Biochem Biophys Res Commun.1988; 156:924–930.CrossrefMedlineGoogle Scholar4 Fratelli M, Gagliardini V, De Blasi A. Low affinity of adrenergic receptors for agonists on intact cells is not due to receptor sequestration. Biochem Biophys Acta.1989; 1012:178–183.CrossrefMedlineGoogle Scholar5 Jetschmann J-U, Benschop RJ, Jacobs R, Kemper A, Oberbeck R, Schmidt RE, Schedlowski M. Expression and in-vivo modulation of α- and β-adrenoceptors on human natural killer (CD16+) cells. J Neuroimmunol.1997; 74:159–164.CrossrefMedlineGoogle Scholar6 Casale TB, Kaliner M. Demonstration that circulating human blood cells have no detectable alpha1-adrenergic receptors by ligand binding analysis. J Allergy Clin Immunol.1984; 742:812–819.Google Scholar7 Maestroni GJ, Conti A. Noradrenergic modulation of lymphohematopoiesis. Int J Immunopharmacol.1994; 16:117–122.CrossrefMedlineGoogle ScholarhypertensionahaHypertensionHypertensionHypertension0194-911X1524-4563Lippincott Williams & WilkinsResponseRicci Alberto, MD, Bronzetti Elena, PhD, Greco Stefania, MD, Conterno Andrea, MD, Mulatero Paolo, MD, Schena Marina, MD, Schiavone Domenica, PhD, Veglio Franco, MD, Tayebati Seyed Khosrow, PharmD, and Amenta Francesco, MD111999A recent paper of our group,R1 reported that human peripheral blood lymphocytes (HPBL) express α1-adrenergic receptor subtypes. Our conclusions are strongly criticized by Drs Kavelaars, Ruppe von der Voort, and Heijnen. Their opinion is that in view of the technical setup of the experiments, it is not demonstrated that freshly isolated HPBL express α1-adrenergic receptors.We do not agree with these criticisms for the reasons detailed below. First, the problem of a rather high density of [3H]prazosin binding sites in HPBL was already discussed in our manuscript. These criticisms were derived mainly from wrong calculations. It is not true that cell lines express at most 4 to 10 times fewer receptors than HPBL in our study. Transfected cell lines were reported to bind α1-adrenergic radioligands in the range between 2856 to 4740 fmol/mg protein.R2R3 The mean protein concentration of preparations of HPBL is 1 mg/2.7 to 3×107 cells.R4 Hence, our preparations of HPBL have an α1-adrenergic receptor concentration similar to that reported for transfected cell lines. Second, references to literature used in our paper were correct with the exception of the quotation of Reference 7 instead of 10 and of Reference 10 instead of 9 in the 2nd paragraph of the Discussion. We were also surprised that in spite of concerns for the presence of α1-adrenergic receptors in HPBL, Kavelaars et al apparently ignored two recent papers,R5R6 which were not published when our work was accepted. In these studies, α1-adrenergic receptors were demonstrated by analyzing lymphocyte catecholamine responseR5 and the amiloride-sensitive sodium channels.R6 Third, in our analysis of [3H]prazosin, binding was performed by using two different approaches: (1) conventional radioligand binding assay with compounds selective for α1-adrenergic receptor subtypes, and (2) antireceptor subtype antibodies. Apparently, the radioligand binding part of the study was not considered by Kavelaars et al. Their criticisms were on experiments with antibodies. Of course, we are unable to establish if and to which extent the antibodies used recognize α1-adrenergic receptor subtypes binding domain. However, the explanation of Kavelaars et al is simplistic as we cannot exclude the occurrence of interactions (indirect) between the antibody and the receptor binding domain after blockade of a portion of receptor sequence with a specific antibody. In line with this hypothesis are binding curves generated in the presence of antibodies. On the other hand, experiments with antibodies are just one of the different approaches for demonstrating α1-adrenergic receptor subtypes. Our assumption of the presence of α1-adrenergic receptor subtypes in HPBL was also confirmed by some recent work using cytospin-centrifuged HPBL and immunocytochemistry. In these experiments, we observed that 25% of HPBL were immunoreactive for α1A receptor protein, 40% for α1B receptor protein, and 22% for α1D receptor protein. Fourth, on the amounts of blood and lymphocytes, Kavelaars et al failed to consider that we collected blood from 30 subjects. We did not use blood from 30 subjects for all experiments; we used a single subject for a series of complete experiments to obtain reliable data for a given parameter. Standard deviations reflect intersubject variability. Fifth, concerning PCR data, note that our protocol did not include quantitative PCR. Lanes show just the presence of signals, whereas quantitative analysis was based on radioligand binding assay data as indicated in our paper. R1 Moreover, in Figure 1 (α1B receptor panel), autoradiography improved detection of a signal affected by interfering factors only in one subject. Really, we do not understand the purpose of these criticisms, which appear inappropriate.In summary, with the exception of some imprecision in quoting references, criticisms of our study by Kavelaars et al do not seem based on justified methodological problems. Our work, as well as more recent evidence,R5R6 indicate that HPBL express α1-adrenergic receptors. A crucial point to investigate in future studies is the significance of α1-adrenergic receptors. 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Forsythe P and Bienenstock J Neural Regulation of the Immune Response Allergy Frontiers: Classification and Pathomechanisms, 10.1007/978-4-431-88315-9_29, (493-514) November 1999Vol 34, Issue 5 Advertisement Article InformationMetrics https://doi.org/10.1161/01.HYP.34.5.e5 Originally publishedNovember 1, 1999 PDF download Advertisement